Promising immunotherapy approaches such as dendritic cell vaccine, cytokine therapy, chimeric antigen receptor (CAR) T cell therapy, checkpoint inhibition, are only as effective as the available T-cells and TAA they target. Potentia TAA candidates must meet a number of criteria: they must be immunogenic, abundantly expressed by tumor cells, expressed less in normal tissue and functionally important for the tumor (so that expression is unlikely to be lost). Mitochondria produce energy in the form of adenosine triphosphate (ATP) via OXPHOS. One byproduct of OXPOS is the production of superoxide radicals or reactive oxygen species (ROS); these and replication errors can damage mtDNA resulting in heteroplasmy associated with sequence mutations. Up to 100% of cancer patients have mutations in their mitochondrial DNA and these Tumor Associated Mitochondria Antigens (TAMAs) meet all of the criteria of an ideal TAA. Mitochondrial DNA encoded proteins are prokaryotic in origin as they have the first methionine methylated and there are 100's to 1000's of copies within a single cell as nucleoids. Thus a propagated mutation with an amino acid substitution could generate novel epitopes comparable to a microbial antigen. To test the hypothesis that TAMAs could be generated a DC vaccine using mitochondria from lysates of RENCA cells, a spontaneously-arising renal adenocarcinoma. DC were pulsed with mitochondria lysate and used to vaccinate mice, both therapeutically and prophylactically, before challenge with RENCA cells. Protective immunity was observed when the vaccine was used prophylactically. It was also effective in delaying tumor growth in a therapeutic setting. We determined that the mitochondrial-encoded cytochrome c oxidase 1 (COX1) and NADH dehydrogenase 5 (ND5) genes were mutated in the RENCA tumor cells used. Immunization of mice with the mutated versions of the peptides impaired RENCA tumor growth in a CD8+ T immunogenic and induce antitumor immune response, we cell-specific manner. Following these exciting results we propose the following Specific Aims: Aim1. To demonstrate hypothesize that the transfer of mutated mitochondria into wild type mitochondria bearing tumor cells lines will induce recognition by TAMAs primed T cells. Aim2. To investigate synergistic effects of TAMA vaccines from heterogeneous mitochondria in combination with immune checkpoint inhibition. PD-L1 is overexpressed in RENCA tumors. To enhance the therapeutic effect of the TAMAs vaccine we will pair PD-1/PD-L1 antagonists with the TAMAs DC vaccine Aim3. To investigate the role of autophagy in TAMA antitumor immune response to mitochondrial heterogeneity in the mouse and human. We hypothesize that autophagy is a relevant mechanism used by tumor cells to remove mutated mtDNA and avoid immune recognition. Its inhibition will enhance immunity. T cell recognition of TAMAs from heterogeneous mitochondria in tumor cybrids.